Prevalence of Parasitic Contamination of Raw Vegetables in Nakhon Si

Punsawad et al. BMC Public Health (2019) 19:34 https://doi.org/10.1186/s12889-018-6358-9

RESEARCH ARTICLE Open Access Prevalence of parasitic contamination of raw vegetables in Nakhon Si Thammarat province, southern Thailand Chuchard Punsawad1,3* , Nonthapan Phasuk1,3, Kanjana Thongtup1, Surasak Nagavirochana1 and Parnpen Viriyavejakul2

Abstract Background: Soil-transmitted helminth (STH) infections are major public health problems in poor and developing countries that require fecal contamination of the environment for transmission. The consumption of raw vegetables without proper washing is one of the main routes of intestinal parasite acquisition. Therefore, this study was designed to detect the prevalence of intestinal parasitic contamination in commonly consumed raw vegetables sold in three central open-air markets in Nakhon Si Thammarat province, southern Thailand. Methods: A total of 265 fresh vegetable samples consisting of peppermint, lettuce, coriander, leek, gotu kola, celery, Chinese cabbage, culantro, Thai basil, and Chinese morning glory were purchased from three central open-air markets in the Mueang, Thasala and Sichon districts from December 2016 to March 2017. Each sample was washed with physiological saline, shaken for 15 min, and then allowed to sediment. Finally, sedimentation was performed via the sedimentation concentration technique and examined using light microscopy for the detection of pathogenic parasites. Results: The overall prevalence of parasitic contamination was 35.1% (93/265). The most predominant parasite was hookworms (42.9%), followed by Strongyloides stercoralis (10.6%), Trichuris trichiura (2.6%), Ascaris lumbricoides (2.6%), and Toxocara spp. (2.6%). The highest level of contamination was found in celery, with a prevalence rate of 63.3% (19/30), while the lowest contamination level was found in Chinese morning glory, with a prevalence rate of 2.0% (2/30). The prevalence of intestinal parasite contamination in Mueang district (51.5%) was significantly higher than that in Thasala district (17.9%) and Sichon district (30.6%) (P < 0.001). Conclusion: The results of the present study demonstrate that consumption of vegetables with parasite contamination in this area represents a potential route for the transmission of parasitic infection, particularly hookworm infection. Therefore, it is necessary for health authorities to educate consumers about the proper washing of vegetables prior to consumption. Preventive methods such as wearing gloves and washing hands after handling vegetables should also be advocated to sellers who are at risk of acquiring STH infections via skin penetration. Keywords: Parasitic contamination, Raw vegetables, Intestinal parasites, Thailand

* Correspondence: [email protected] 1School of Medicine, Walailak University, 222 Thasala district, Nakhon Si Thammarat 80161, Thailand 3Tropical Medicine Research Unit, Research Institute for Health Sciences, Walailak University, 222 Thasala district, Nakhon Si Thammarat 80161, Thailand Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Punsawad et al. BMC Public Health (2019) 19:34 Page 2 of 7

Background 45′6″E longitude. The average annual temperature is Parasitic infections represent a major global public health 28.2 °C, and approximately 1702.6 mm of precipitation problem. The burden of parasitic infections often affects falls annually (Climatological Center, Thai Meteoro- developing countries, which frequently lack good sanitiza- logical Department, Annual report 2015). According to tion and personal hygiene practices. Moreover, environ- official statistic registration systems, the total population mental factors such as climate, geography, temperature, in this area is approximately 1,557,482. Fresh vegetable soil type, and rainfall also play important roles that con- samples were purchased from three central open-air tribute to the prevalence of parasitic infections. markets located in Mueang (the capital district of In southern Thailand, the most prevalent parasitic Nakhon Si Thammarat province), Thasala (approxi- infection among all age groups has been soil-transmitted mately 30 km away from the capital district) and Sichon helminth (STH) infections, especially hookworm infec- districts (approximately 70 km away from the capital dis- tion [1–4], which may transmit to humans through ei- trict) (Fig. 1). The fresh vegetables sold in these markets ther skin penetration or the ingestion of its larvae [5]. were brought from different farms and agricultural areas The consumption of fresh vegetables is a common in different parts of Nakhon Si Thammarat province. eating habit among people in southern Thailand. Fur- ther, healthy food trends in Thailand are now focused on Sample collection sufficient daily vegetable consumption, as this could help A total of 265 fresh vegetable samples including 10 differ- prevent major diseases, such as cardiovascular disease ent types that are frequently consumed without cooking and certain cancers. The WHO also recommended the were randomly purchased from sellers in three central intake of a minimum of 400 g of fruit and vegetables per open-air markets. The fresh raw vegetable samples used in day for micronutrient supplementation as well as the this study included peppermint (Mentha x piperita), let- prevention of the aforementioned chronic diseases [6]. tuce (Lactuca sativa), coriander (Coriandrum sativum), However, eating raw vegetables may also lead to the leek (Allium porrum), gotu kola (Centella asiatica), celery transmission of certain human pathogens (fresh fruits (Apium graveolens), Chinese cabbage (Brassica rapa and vegetables are vectors for the transmission of human pathogens) [7]. Several studies have reported the parasitic contamination of fresh vegetables in many countries around the world, for example, Arba Minch town, southern Ethiopia [8], Khartoum state, Sudan [9], Benha, Egypt [10], Accra, Ghana [4], Mazandaran province, northern Iran [11], Poland [12], and Metro Manila, Philippines [13]. Parasites and protozoa that were common contaminants included Ascaris lumbricoides, Crypto- sporidium spp., Entamoeba histolytica/dispar, Enterobius vermicularis, Giardia intestinalis,hookworm,Hymenole- pis spp., and Trichuris trichiura [4, 8–12, 14]. These contaminants could result from water used to moisten vegetables and postharvesting handling methods [8, 9, 15]. Nakhon Si Thammarat province is an endemic area for STH infection [3]. To date, there have been no reports on parasitic vegetable contamination in this area. Therefore, the main aim of this study was to determine the prevalence of parasitic contamination in commonly consumed vegetables in Nakhon Si Thammarat province, southern Thailand.

Methods Study area A cross-sectional study was conducted from December 2016 to March 2017 in Nakhon Si Thammarat province, which is located in southern Thailand approximately Fig. 1 Location of the study area including the Mueang (1), Thasala (2) 800 km from Bangkok, the capital of Thailand. This area and Sichon districts (3) of Nakhon Si Thammarat province, southern has a tropical rainforest climate and is located at the Thailand (Map from Wikimedia Commons: https://commons.wikimedia. org/wiki/File:Amphoe_8001.svg) geographical coordinates of 8°43′10″N latitude and 99° Punsawad et al. BMC Public Health (2019) 19:34 Page 3 of 7

subsp. pekinensis), culantro (Eryngium foetidum), Thai rate of parasitic contamination among different types of basil (Ocimum basilicum), and Chinese morning glory vegetable and among different markets. A p-value less (Ipomoea aquatica). The pictures of vegetable samples than 0.05 was considered statistically significant. were demonstrated in Fig. 2. The fresh vegetable samples were collected in clean, labeled plastic bags and trans- Results ported immediately to the parasitology laboratory at the A total of 265 fresh vegetable samples were examined School of Medicine, Walailak University for parasitic for the presence of parasite contamination. The results examination. of parasitic contamination in vegetable samples are shown in Table 1. The parasites detected in vegetable Detection of intestinal parasites samples were hookworm, Strongyloides stercoralis, Tri- Fresh vegetable samples weighing 200 g were washed churis trichiura, Ascaris lumbricoides, and Blastocystis with 1000 mL of physiological saline solution (0.9% so- spp. Pictures of some of the parasites found in this dium chloride) and shaken for 15 min in order to separ- survey were demonstrated in Fig. 3. The overall rate of ate the parasites from vegetables. Then, the washing intestinal parasite detection was 35.1% (93/265) in all water was collected and left overnight to allow sedimen- vegetable samples. The highest rate of contamination tation. Afterward, the supernatant was decanted, and the was found in celery [63.3% (19/30)] while the lowest was remaining washing water was transferred to 12 mL con- found in Chinese morning glory [6.8% (2/30)] (Table 1). ical tubes. To concentrate the parasitic stages, the sedi- The highest rate of hookworm contamination was found ment was centrifuged at 2000×g for 15 min. After in celery, but not Thai basil or Chinese morning glory. centrifugation, the supernatant was carefully removed Strongyloides larvae were most frequently detected in without shaking. Then, the sediment was agitated gently celery but in lettuce. Eggs of Trichuris trichiura were and examined under a light microscope using 10× and frequently detected in culantro but were not found in 40× objectives. To increase the chance of parasite detec- peppermint, lettuce, coriander, leek, gotu kola, or celery. tion, three slides were prepared from each sample by Eggs of Ascaris lumbricoides were frequently detected in two independent investigators. lettuce, coriander, and celery. Table 2 shows the prevalence of parasitic contamin- Statistical analysis ation in different vegetable samples among the three Data analysis was performed with IBM SPSS Statistics markets. The parasites were detected in 51.5%, 30.6%, for Windows, Version 23.0. Armonk, NY: IBM Corp. and 17.9% of samples obtained from Mueang, Thasala, Qualitative variables were described by frequency (per- and Sichon districts, respectively. The highest rate of centage). A chi-squared test was used to compare the parasitic contamination in vegetables was found in

Fig. 2 The pictures of vegetable samples including a. peppermint (Mentha x piperita), b. lettuce (Lactuca sativa), c. coriander (Coriandrum sativum), d.leek(Allium porrum), e.gotukola(Centella asiatica), f.celery(Apium graveolens), g. Chinese cabbage (Brassica rapa subsp. pekinensis), h.culantro (Eryngium foetidum), i. Thai basil (Ocimum basilicum), and j. Chinese morning glory (Ipomoea aquatica) Punsawad et al. BMC Public Health (2019) 19:34 Page 4 of 7

Table 1 Distribution of intestinal parasites in fresh vegetable samples collected from three markets in Nakhon Si Thammarat province, Thailand Vegetables Number of Hookworm Strongyloides Trichuris trichiura Ascaris lumbricoides Toxocara spp. Number sample Eggs (%) stercoralis larvae eggs eggs eggs positive (%)a Peppermint 15 6 (40.0) 2 (13.3) 0 0 1 (6.7) 9 (60.0) Lettuce 20 1 (5.0) 0 0 3 (15) 0 4 (20.0) Coriander 29 8 (27.6) 2 (6.9) 0 3 (10.3) 0 13 (44.8) Leek 30 7 (23.3) 6 (20.0) 0 0 0 13 (43.3) Gotu kola 21 7 (33.3) 4 (19.0) 0 0 1 (4.8) 12 (57.1) Celery 30 9 (30.0) 9 (30.0) 0 1 (3.3) 0 19 (63.3) Chinese cabbage 30 2 (6.7) 1 (3.3) 2 (6.7) 0 2 (6.7) 7 (23.3) Culantro 30 4 (13.3) 2 (6.7) 3 (10.0) 0 2 (6.7) 11 (36.7) Thai Basil 30 0 1 (3.3) 1 (3.3) 0 1 (3.3) 3 (10.0) Chinese morning glory 30 0 1 (3.3) 1 (3.3) 0 0 2 (6.7) Total 265 44 (16.6) 28 (10.6) 7 (2.6) 7 (2.6) 7 (2.6) 93 (35.1) a Number of positive parasitic contamination events are significantly different among types of vegetables (P < 0.001)

Mueang district, whereas the lowest rate was observed understand the potential source of pathogenic parasite in Thasala district. Statistical analysis revealed that the acquisition in this study area. This study demonstrated rate of contamination in vegetables obtained from that the prevalence of parasitic contamination in Mueang district was significantly higher than those ob- vegetable samples from Nakhon Si Thammarat prov- tained from Sichon and Thasala district (P < 0.001). ince was 35.1%, which was comparable to the prevalence of 32.6% and 34.7% from previous reports in Discussion Shahrekord, Iran [16] and Poland [17], respectively. The consumption of raw vegetables plays an important However, higher contamination rates, ranging from role in the transmission of parasites to humans. The 45% to 58%, were detected in Libya, Iran, Egypt, recovery of parasites from vegetables helped us better Brazil, and the Philippines [8, 13, 18–22]. In contrast,

Fig. 3 Representative images of parasites found in this survey. Hookworm egg (a), Trichuris trichiura egg (b), Ascaris lumbricoides eggs (c) and Strongyloides stercoralis larvae (d). Original magnification: 40X Punsawad et al. BMC Public Health (2019) 19:34 Page 5 of 7

Table 2 Distribution of intestinal parasite contamination in different fresh vegetables among the three markets in Nakhon Si Thammarat province, Thailand Vegetable type No. of examined Mueang district Thasala district Sichon district samples Examined Positive (%) No. of No. of positive No. of No. of positive No. of No. of positive examined (%) examined (%) examined (%) Peppermint 15 9 (60.0) 11 7 (63.6) 0 0 (0.0) 4 4 (40.0) Lettuce 20 4 (20.0) 10 4 (40.0) 6 0 (0.0) 4 0 (0.0) Coriander 29 13 (44.8) 10 8 (80.0) 9 1 (11.1) 10 4 (40.0) Leek 30 13 (43.3) 10 6 (60.0) 10 4 (40.0) 10 3 (30.0) Gotu kola 21 12 (57.1) 10 7 (70.0) 3 2 (66.7) 8 3 (37.5) Celery 30 19 (63.3) 10 9 (80.0) 10 4 (40.0) 10 6 (60.0) Chinese cabbage 30 7 (23.3) 10 3 (30.0) 10 1 (10.0) 10 3 (30.0) Culantro 30 11 (36.7) 10 5 (50.0) 10 1 (10.0) 10 5 (50.0) Thai Basil 30 3 (10.0) 10 2 (20.0) 10 0 (0.0) 10 2 (20.0) Chinese morning glory 30 2 (6.7) 10 1 (10.0) 10 1 (10.0) 10 0 (0.0) Total 265 93 (35.1) 101 52 (51.5)a 78 14 (17.9)a 85 26 (30.6)a a Number of positive parasitic contamination events are significantly different among three markets (P < 0.001) the rate of contamination was lower in some studies, such roots to stalks. In contrast, the lowest parasitic contam- as 5.9% in Ankara, Turkey [23], 14.89% in Mazandaran ination was observed in Chinese morning glory, which province, northern Iran [11], 19.4% in Alexandria, Egypt could be because the smooth surface of its stalks that [24], and 26% in Hanoi, Vietnam [25]. According to the may reduce the probability of parasitic attachment. influence of season on the prevalence of parasites, previ- In this present study, hookworm was detected in ous studies reported that the rate of STH contamination 16.60% (44/265) of vegetables examined and was the was higher in warm seasons versus cold seasons [11, 19, most predominant pathogenic parasite. This finding is in 26]. This finding suggested that the transmission and agreement with other studies conducted in Ghana (13%) prevalence rate of parasites were associated with climate [4], Sudan (5.7%) [9], and northern Iran (4.40%) [11]. In and temperature. Differences between this study and contrast, no ova of hookworm species were recovered in others might be attributed to variations in climatic condi- some previous studies [8, 10, 16, 18–20, 22–25, 27]. Our tions, types of soil, types of water used for agriculture, and previous report in 2017 demonstrated that hookworm poor hygienic practices during the transportation and infection was the most prevalent parasitic infection in marketing of vegetables. Nopphitam district, Nakhon Si Thammarat province, Celery was found to be the most frequently contami- southern Thailand [2]. The high prevalence of hook- nated sample (63.3%) followed by peppermint (60.0%), worm egg contamination of vegetables in this study gotu kola (57.1%), coriander (44.8%), leek (43.3%), culan- could be attributed to poor sanitation and the use of tro (36.7%), Chinese cabbage (23.3%), lettuce (20%), and human waste-contaminated water for irrigation in the Thai basil (20%), whereas Chinese morning glory (6.7%) region. Lack of proper footwear and exposure of skin to was the least contaminated. These results were different contaminated soil might be responsible for hookworm from previous studies, which reported that lettuce pos- infection in the study area. In addition, the high preva- sessed the highest parasitic contamination [9, 10, 21]. In lence of hookworm might be due to differences in geo- Thailand, celery is commonly used for seasoning and graphical location, climate conditions, and the types of garnishing in many Thai dishes. The highest contamin- soil [28, 29]. The second most prevalent contamination ation in celery might be because celery consists of a found in this study was the larvae of Strongyloides ster- number of stalks that are connected at the base with its coralis. Our finding was consistent with previous studies leaves near the top of stalks. The structure of its stalk is in Accra, Ghana and in Jimma Town, Ethiopia, which roughly U-shaped in a slit pattern, which allows the reported a 43% and 21.9% prevalence of Strongyloides parasites to attach more easily to the surface of these stercoralis contamination, respectively [4, 22]. A high vegetables and makes them more difficult to remove. rate of Strongyloides contamination might be because Normally, celery samples obtained from the three mar- Strongyloides spp. has a complex life cycle with a kets were frequently sold with roots and stalks, which free-living stage in the environment that does not re- might increase the chance of soil contamination from quire a host for its proliferation [30]. Punsawad et al. BMC Public Health (2019) 19:34 Page 6 of 7

Aside from the contamination of hookworm eggs, T. helminths in raw vegetables [16, 23, 33]. Furthermore, to trichiura and Ascaris lumbricoides eggs were also de- emphasize on the proper washing procedure, the previous tected in the vegetable samples collected from this area. study in Iran demonstrated that the pre-washing proced- Trichuriasis occurs by ingestion of contaminated food ure using tap water or underground water could not com- and water with embryonated eggs of T. trichiura [31]. In pletely get rid of parasites from vegetables [26]. Hence, the present study, eggs of T. trichiura were detected in the health authorities should provide the knowledge of 2.64% (7/265) of vegetable samples. This finding was proper washing method for local people in order to pre- consistent with previous reports from Khartoum state, vent parasitic transmission. Sudan [9], Mazandaran province, northern Iran [11], However, it is important to note that our study has Accra, Ghana [4] and in villages of Qazvin province, Iran several limitations. This study did not demonstrate the [27], where the contamination rates were 2.9, 2.2, 2 and effect of seasonal variation on parasitic contamination. 0.9%, respectively. In addition, eggs of Ascaris lumbri- We did not address the intensity of vegetable washing coides were detected in 2.64% (7/265) of vegetable before display for sale or the source of water used by samples. The rate of contamination with A. lumbricoides each seller. These factors might affect the rate of vege- eggs was 68% in Tripoli, Libya [18], 20.83% in Arba table contamination in our study. Minch town, southern Ethiopia [8] and 8.17% in Shahre- kord, Iran [26]. Contamination by these STHs in vege- Conclusion table samples might occur at any point along the chain; The results of the present study indicate that the con- during planting, harvesting, transportation, or the mar- tamination of raw vegetables with pathogenic parasites keting of vegetables. The differences in the degree of in Nakhon Si Thammarat province, southern Thailand contamination might be attributed to the types of soil, might represent a transmission vector for intestinal par- the quality of water used for planting and irrigation, and asites to consumers. Prevention methods such as proper hygienic practices during the process of marketing. washing or cooking of vegetables before consumption Beside STH contamination, this study also detected should be conveyed to consumers. In addition, compre- Toxocara spp. eggs in 2.64% (7/265) of fresh vegetable hensive health education and hygienic practices, includ- samples. In other locales, the rate of contamination with ing wearing gloves and washing hands after handling Toxocara spp. eggs in vegetables was 37% in Libya [18], vegetables, should be provided to sellers and farmers. 15.83% in Arba Minch town, southern Ethiopia [8], 3.3% in Shahrekord, Iran [16], 3% in Hanoi, Vietnam [25], and Abbreviations STH: Soil-transmitted helminth; WHO: World Health Organization 1.5% in Ankara, Turkey [23]. In contrast, no ova of Toxo- cara spp. in vegetables were reported in Ardabil, Khorra- Acknowledgements mabad, and Qasvin, Iran, Accra, Ghana, Benha and The authors wish to thank the vegetable sellers who participated in the study. The authors would like to acknowledge Dr. Prachyapan Petchuay, MD, Alexandria, Egypt, Minas Gerais, Brazil, or Khartoum PhD, Dean School of Medicine, Walailak University for their support state, Sudan [4, 9, 10, 19–21, 24, 27]. Human toxocariasis throughout the course of this study. We also thank Miss Potiga Chotipong is a helminthic zoonosis caused by larval stages of Toxo- for preparing reagents and laboratory instruments. cara canis and less frequently by Toxocara cati [32]. The Funding long term survival of Toxocara spp. outside their hosts This study was partially financially supported by the Tropical Diseases and coupled with their high fecundity is responsible for signifi- Parasitic Infectious Diseases Research Group (WU60512), granted by the Institute of Research and Development, Walailak University. The funder cant contamination of soil with infective eggs [32]. This played no role in the study design, data collection, and data analysis and study indicates that domestic animals (dogs and cats), interpretation. which are the source of Toxocara eggs, may at some point shed contaminated feces onto cultivation areas. Availability of data and materials All data generated or analyzed during this study are available from the The parasitic contamination rates were significantly corresponding author on reasonable request. different for samples collected from different markets; samples collected from Muang district showed the high- Authors’ contributions KT, SN, NP, PV, and CP conceived and designed the study. KT, SN, NP, and CP est rate of contamination. The differences between the performed data collection. KT, SN, NP, PV, and CP performed the experiments. three markets might be due to the different sources of KT, NP, PV, and CP analyzed the data. NP, PV, and CP wrote the manuscript. All vegetables as well as the hygienic practices in handling authors read and approved the final manuscript. and washing by different sellers. Ethics approval and consent to participate The results of this study emphasized that raw vegetables This study protocol was reviewed and approved by the Ethics Committee on from the markets in the study area could be possible vehi- Human Rights Related to Research Involving Human Subjects, Walailak cles of parasitic transmission to humans. The previous University prior to the collection of samples (Protocol No. WU-EC-MD-4-118-59). studies revealed that the standard washing procedure was Consent for publication an effective method to prevent the contaminations of Not applicable. Punsawad et al. BMC Public Health (2019) 19:34 Page 7 of 7

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